Thr4 phosphorylation on RNA Pol II occurs at early transcription regulating 3’-end processing

RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on Ser2/Ser5 of the C-terminal heptad repeats is well-established, Thr4’s role remains enigmatic. Paradoxically, Thr4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and mRNA processing. Our investigation revealed that Thr4 phosphorylation detection was obstructed by flanking Ser5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via Thr4 phosphorylation, which previously were attributed to Ser2. Importantly, loss of Thr4 phosphorylation greatly reduces Ser2 phosphorylation, revealing a crosstalk between the two marks. Finally, the function analysis of the Thr4 phosphorylation highlighted its role in alternative 3’-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the CTD.